Historical introduction, overview, and reproductive biology of the ...

1
REVIEW / SYNTHÈSE
Historical introduction, overview, and reproductive
biology of the protochordates 1
Charles C. Lambert
Abstract: This issue of the Canadian Journal of Zoology exhaustively reviews most major aspects of protochordate biology
by specialists in their fields. Protochordates are members of two deuterostome phyla that are exclusively marine.
The Hemichordata, with solitary enteropneusts and colonial pterobranchs, share a ciliated larva with echinoderms and
appear to be closely related, but they also have many chordate-like features. The invertebrate chordates are composed
of the exclusively solitary cephalochordates and the tunicates with both solitary and colonial forms. The cephalochordates
are all free-swimming, but the tunicates include both sessile and free-swimming forms. Here I explore the history
of research on protochordates, show how views on their relationships have changed with time, and review some of
their reproductive and structural traits not included in other contributions to this special issue.
Résumé : Le numéro courant de la Revue canadienne de zoologie présente une synthèse exhaustive par des spécialistes
des principaux aspects de la biologie des protochordés. Les protochordés appartiennent à deux phylums exclusivement
marins de deutérostomiens. Les hémichordés qui contiennent les entéropneustes solitaires et les ptérobranches coloniaux
ont, comme les échinodermes, des larves ciliées et semblent leur être apparentés, tout en ayant de nombreuses caractéristiques
semblables à celles des chordés. Les chordés invertébrés comprennent les céphalochordés qui sont toujours solitaires
et les tuniciers qui possèdent des formes solitaires et coloniales. Les céphalochordés nagent librement dans
l’eau, alors que les tuniciers sont représentés par des formes nageuses et sessiles. Ma contribution retrace l’histoire de
la recherche sur les protochordés, elle montre comment les relations entre les groupes ont été interprétées différemment
au cours des années et elle fait le point sur certaines caractéristiques reproductives et structurales qui ne sont pas traitées
dans les autres présentations de cette série.
[Traduit par la Rédaction] Lambert 7
Historical overview
This issue of the Canadian Journal of Zoology is devoted
to protochordates, an intriguing and somewhat disparate
group of marine invertebrates. These reviews convey the
excitement of the current renaissance of interest in these
members of two deuterostome phyla: Hemichordata and the
invertebrate members of the Chordata. Molecular data places
the hemichordates closer to the echinoderms than the chordates,
but we treat the groups here as they have been considered
historically. In this issue, Ruppert (2005) critically considers
the relationship between these organisms in morphological
Received 5 August 2004. Accepted 17 November 2004.
Published on the NRC Research Press Web site at
http://cjz.nrc.ca on 15 April 2005.
C.C. Lambert. 2 University of Washington Friday Harbor
Laboratories, Friday Harbor, WA 98250, USA (e-mail:
clambert@fullerton.edu).
1 This review is one of a series dealing with aspects of the
biology of the Protochordata. This series is one of several
virtual symposia on the biology of neglected groups that will
be published in the Journal from time to time.
2 Mailing address: 12001 11th Avenue NW, Seattle,
WA 98177, USA.
terms, while Zeng and Swalla (2005) examines their molecular
phylogeny and suggests that tunicates be placed in their
own phylum. The designation hemichordate was by Bateson
(1885), who considered their development sufficiently
chordate-like to place them in the same phylum as the other
chordates, but Hyman (1959) and most modern authors regard
them as a separate phylum. There are fewer than 100
species of hemichordates, which include the solitary class
Enteropneusta with about 70 species of benthic organisms
living in soft substrates and the class Pterobranchia with
only 21 species of tubiculous and colonial organisms living
on shells and other hard substrates (Ruppert and Barnes
1994).
The invertebrate chordates include two subphyla: Tunicata
and Cephalochordata. Since the cephalochordates lack the
most anterior portions of the vertebrate nervous system, the
term Acrania has also been applied to them. The cephalochordates
are a small group with only about 30 species of
free-living benthic organisms that live in coarse sands
throughout the world (reviewed by Lambert 2005). They
were considered mollusks until their fish-like nature was discovered
in the 19th century. The Tunicata (often incorrectly
called the Urochordata; P. Kott, personal communication)
are the most prevalent protochordates and include both freeswimming
and fixed classes. With few exceptions, they are
Can. J. Zool. 83: 1–7 (2005) doi: 10.1139/Z04-160 © 2005 NRC Canada

2 Can. J. Zool. Vol. 83, 2005
all hermaphrodites. Thaliaceans and appendicularians (sometimes
called larvaceans) are free-swimming, whereas ascidians
and sorberaceans (Monniot and Monniot 1990; but see
Kott 1989b, 1998) are generally anchored as adults.
Thaliaceans include pyrosomid colonies as well as solitary
and colonial doliolids and salps. Life cycles reach extremes
of complexity in this group. Like the thaliaceans, appendicularians
are also planktonic but lack colonial forms
and have simple life cycles (Ruppert and Barnes 1994). Although
pelagic tunicates include relatively few species, their
biomass can be quite high and they occupy important places
in marine food webs (for review and literature see Bone
1998). Ascidians are convenient to collect and very useful
experimental material; consequently, they are the best
known protochordates and as a result the majority of this
overview deals with them. Lambert (2005) examines the
ecology and natural history of protochordates, which is the
first comprehensive review of the field in over 30 years
(Millar 1971).
Ascidians are generally sessile with no resemblance to
vertebrates, but their larvae have chordate traits, including a
notochord, dorsal tubular nerve cord, and pharyngeal gill
clefts. They constitute the most numerous class of tunicates.
Because they are mostly sessile as adults, through history
they have been variously classified as plants or animals
(Berrill 1950). Aristotle recognized their animal affinities,
and they were assigned to various groups as science progressed
and techniques for observation became more acute.
Mainly, the colonial forms were considered to be cnidarians
that were related to corals (Monniot et al. 1991). Linnaeus in
1767 placed the solitary ascidians in the genus Ascidia and
described Ascidia intestinalis, which now is in the genus
Ciona and is probably the world’s best known ascidian.
Lamarck (1816) named the group Tunicata, and Cuvier
(1815) added much to our knowledge of their anatomy but,
along with many others, classified them with lamellibranch
mollusks, an affinity that was endorsed as late as 1905
(Alder and Hancock 1905–1907). Savigny (1816) clearly
showed the relationship between solitary and colonial ascidians
and also showed that they were not allied with the
lamellibranch mollusks. Through most of the remainder of
the 19th century there were numerous publications on the
structure, larvae, and metamorphosis of ascidians, but no
one had pointed out their obvious chordate nature. In 1867,
Kowalevsky published the detailed development and structure
of the eggs and larvae of Ciona intestinalis and
Phallusia mammillata and pointed out their affinity with the
Chordata. This finding electrified Darwin as well as Huxley
and other champions of Darwin’s Origin of the Species By
Means of Natural Selection, or the Preservation of Favoured
Races in the Struggle for Life. A paradigm shift was required
to accept that tunicates were chordates, not mollusks, and
such a shift required time as many workers who considered
them mollusks were replaced by those who agreed with
Kowalevsky.
During the 19th century numerous coastal areas were extensively
explored and many ascidians described, which resulted
in many fine taxonomic publications. As more species
were described and details of their anatomy were revealed, it
became necessary to erect classification schemes to better
enable scientists to categorize taxa into manageable groups.
Foremost in these efforts was the work of Lahille (1886)
that proposed three orders based upon the structure of the
branchial sac: Aplousobranchia, Phlebobranchia, and Stolidobranchia.
Many of us today still prefer this categorization.
In 1898, Perrier recommended another scheme based
upon the position of the gonads. The Enterogona included
Lahille’s Aplousobranchia and Phlebobranchia and the
Pleurogona included only the Stolidobranchia. Subsequently,
many workers adopted Perrier’s classification, with Lahille’s
orders becoming suborders (Berrill 1950). However, this
idea implies that the aplousobranchs are more closely related
to the phlebobranchs than either group to the stolidobranchs.
Until such time as this is verified by molecular or other
means, it would be prudent to continue to apply Lahille’s orders,
especially since they have precedence.
Increasing interest in the structure, development, physiology,
and biogeography of ascidians led to several valuable
books and major monographs on all aspects of their lives.
Herdman (1882, 1886) admirably reviewed the major works
of the 19th century. Huus (1937) reviewed many articles that
dealt with all aspects of ascidians, including their structure,
development, biogeography, and classification. Brien (1948)
reviewed structure and reproduction. Berrill (1950) continues
to be a widely used classic reference on all aspects of
ascidians with a description of the British species; his subsequent
book in 1955, The Origin of Vertebrates, though still
useful contains some ideas that are no longer accepted. The
Monniots published a superb overview of the class as part of
their account of tropical ascidians (Monniot et al. 1991). The
Biology of Ascidians (Sawada et al. 2001) with a collection
of 69 papers on all aspects of ascidians appeared in 2001.
In addition to these overview accounts, more specialized
reviews have emphasized specific attributes of the protochordates.
Several aspects of the biology of the group appeared
in Protochordates (Barrington and Jefferies 1975).
Millar (1971) reviewed ecology and Goodbody (1974) reviewed
ascidian physiology. Satoh (1994), and later, Jeffery
and Swalla (1997) reviewed the extensive literature on development
and Whittaker (1997) reviewed cephalochordates
in the same volume. A special issue of the American Zoologist
contained a series of papers on ascidian development in
1982. The recent review by Burighel and Cloney (1997) admirably
summarizes ascidian structure and function.
Fertilization and development
Many hemichordates produce a swimming larva similar to
an echinoderm larva, one of the similar traits between hemichordates
and echinoderms. Enteropneusts are exclusively
dioecious, but pterobranch colonies can include both sexes.
Hemichordates from both classes show extensive asexual development
(Hadfield 1975). Spawning is apparently epidemic
and related to tides or temperature (for a review see Hadfield
1975).
Cephalochordates are usually dioecious but may have
skewed sex ratios in nature (Kubokawa et al. 2003). They
are incapable of budding or asexual development, but larvae
may become sexually mature if blocked from metamorphosis
(Wickstead 1975). Whittaker (1997) has thoroughly reviewed
their development.
© 2005 NRC Canada

Lambert 3
In this issue, Bates (2005) discusses environmental signals
and reproductive strategies in ascidians, while here I focus
on spawning, fertilization, and development. Light is an
important cue in ascidian spawning. Some, such as the phlebobranchs
C. intestinalis (Lambert and Brandt 1967;
Whittingham 1967), Corella parallelogramma (Huus 1939),
Corella inflata, and Corella willmeriana (Lambert et al. 1981),
spawn within a few minutes of light following darkness.
Spawning requires a longer light exposure in several stolidobranchs
(Rose 1939; West and Lambert 1976; Numakunai and
Hoshino 1980; Jeffery 1990). However, another stolidobranch,
Molgula manhattensis, requires only a short exposure
(Whittingham 1967). Interestingly, C. intestinalis populations
from different regions have dissimilar responses to
light. This species from the Atlantic and Pacific US coasts
spawns in light following darkness (Lambert and Brandt
1967; Whittingham 1967), but the same species spawns in
response to darkness following light in the Mediterranean
(Georges 1968; C.C. Lambert, unpublished observation). The
target for light reception is included in the gonoducts and
dorsal strand of C. intestinalis (Reese 1967; Woollacott 1974)
and an action spectrum for spawning suggests a cytochrome
for the receptor molecule (Lambert and Brandt 1967).
Like many sessile organisms all ascidians are hermaphrodites,
although many species are protandrous (Sabbadin et
al. 1991). Most solitary ascidians spawn their eggs freely
into the sea where fertilization and development take place,
but a few species brood within the atrium (Lambert et al.
1995). All colonial forms brood their eggs, either in special
brood chambers or in the atrium. Sperm is shed into the sea
and some colonial forms can store exogenous sperm for prolonged
periods (Bishop and Ryland 1991).
A tadpole larva, which may swim for 12 h to several days,
is produced by most free-spawning species; however, larvae
from brooded eggs generally swim for only a few minutes or
hours (Lambert 1968; Lambert et al. 1995). In this issue,
McHenry (2005) reviews swimming of ascidian larvae, and
Lambert (2005) and Bates (2005) consider the ecological
consequences of a shortened swimming period. Still another
reproductive strategy is seen in several Molgula species
where the spawned egg develops directly into a functional
juvenile without going through a swimming larval stage
(Berrill 1931; Bates 2005). Tadpole release from the colony
appears to be under the control of light (Watanabe and Lambert
1973; Forward et al. 2000.). Light and gravity also influence
the behavior of swimming larvae. The review by McHenry
(2005) expands on the behavior of larval ascidians.
Ascidian eggs are enclosed within cellular and noncellular
vestments. A layer of test cells surrounds the egg and contributes
to the larval tunic (Cloney and Hansson 1996;
Takamura et al. 1996). These cells are enclosed within a
noncellular vitelline coat (termed chorion in older literature).
Around the outside of the vitelline coat (VC) is a single
layer of follicle cells that function in protein and RNA synthesis
(Jeffery 1980), egg flotation (Lambert and Lambert
1978), and egg adhesion (Young et al. 1988; Lambert et al.
1995) in some species. They are required for maturation in
Halocynthia roretzi (Sakairi and Shirai 1991), fertilization
(Hoshi et al. 1981; Hice and Moody 1988; Fuke and
Numakunai 1996), and the block to polyspermy in others
(Lambert et al. 1997). They also contribute to the block to
self-fertilization in C. intestinalis (Marino et al. 1999) and
H. roretzi (Fuke and Numakunai 1996). This layer of cells is
the inner of two follicle cell layers present in the ovarian
follicle (Burighel and Cloney 1997). Ascidian eggs have at
least two blocks to polyspermy: the follicle cell-generated
fast block and a slower electrical block (Lambert et al.
1997). Polyspermy blocks are essential for normal development,
as ascidians often live in massive aggregations and the
sperm concentration when the eggs are released may be
quite high.
Solitary ascidians generally produce a large number of
eggs that are around 150 µm in diameter, whereas compound
forms usually have only a few but much larger eggs. Forms
with external fertilization like most phlebobranchs and
stolidobranchs spawn small, rather simple sperm; those with
internal fertilization like all aplousobranchs have larger and
more complex sperm (Lambert 1982; Martinucci et al.
2001). Sperm from solitary ascidians can swim for extended
periods and undergo hypermotility in the vicinity of conspecific
eggs (Bolton and Havenhand 1996). In addition,
ascidian eggs release factors that increase the motility of
sperm and can cause the directed swimming of the sperm to
the egg (Miller 1975; Morisawa et al. 2001; Yoshida et al.
2002; Ishikawa et al. 2004).
Sperm of hemichordates, cephalochordates, and appendicularians
have a large apical acrosome and a midpiece posterior
to the nucleus. During fertilization an acrosomal filament
extends and fuses with the egg (for a review see Jamieson
1991). In contrast, the sperm of ascidians and thaliaceans
have a much reduced acrosome and lack a classical midpiece.
The single mitochondrion is adjacent to the nucleus in
the head (Retzius 1904, 1905; Franzén 1976;Villa 1977).
During fertilization the mitochondrion slides down the tail
by an actin–myosin driven process (Lambert and Lambert
1984) as the sperm passes through the VC (Lambert and
Epel 1979; Lambert and Lambert 1983; Lambert 1989; Lambert
and Battaglia 1993).
All ascidians are hermaphrodites, but many cannot fertilize
their own eggs. For instance C. intestinalis, Ciona savignyi,
and all pyurids such as Halocynthia and Pyura species
are generally self-sterile, whereas many other species including
many styelids and most ascidiids are self-fertile, at least
in the laboratory. The mechanism of this specificity involves
a particular protein in the VC (Fuke and Numakunai 1996,
1999; Marino et al. 1998, 1999; Sawada and Yokosawa
2001). Removal of the VC allows self-fertilization (Byrd and
Lambert 2000). The sperm binds to the egg through a sperm
surface glycosidase binding to a VC glycoside (Hoshi 1986;
Lambert and Koch 1988; Honegger 1992) or through a
sperm proteasome binding to extra cellular ubiquitin
(Sawada et al. 2002). Fertilization is species-specific as long
as the VC is present (Reverberi 1971; Patricolo and Villa
1992), but sperm interaction with follicle cells is not. This
can lead to interspecific sperm competition: sperm from Ascidia
sydneiensis and Phallusia julinea interfere with the fertilization
of Phallusia nigra eggs (Lambert 2000). The block
to self-fertilization in both C. intestinalis and C. savignyi is
removed by acid treatment, but this does not allow interspecific
hybridization (Byrd and Lambert 2000).
Interspecific fertilization is generally not possible, but it is
possible to obtain fertilization between two Molgula species
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4 Can. J. Zool. Vol. 83, 2005
in which one has a tailed larva and the other lacks a tail. In
this cross, the molecular switches to anural development
have been discovered (Swalla and Jeffery 1990; Swalla et al.
1999).
Following fertilization, the egg experiences multiple
pulses in intracellular calcium emanating from the fertilization
site (Roegiers et al. 1995; Dumollard et al. 2004), leading
to cortical and endoplasmic reticulum reorganization in
the zygote (Speksnijder et al. 1995). Conklin (1905a) was
the first to note the pigmented plasms present in the zygotes
of Styela canopus (formerly Cynthia partita). He found that
the plasms became localized in distinct parts of the tadpole
larva and was able to determine the cell lineages involved.
Subsequently, pigmented plasms have been seen in eggs of
Boltenia villosa, Styela plicata, and a few other species;
however, most ascidian zygotes and embryos lack such
markers. Conklin (1905b) went on to confirm, by blastomere
deletion studies, that ascidian eggs were strictly mosaic and
only right or left half larvae were produced from one of the
first two blastomeres, a finding that has been verified by
many other studies with various species. This is an intriguing
result in view of the tremendous regenerative powers of adult
ascidians. Indeed, half embryos can metamorphose into fully
functional adults with all structures present (Nakauchi and
Takashita 1983). The numerous studies implicating mosaic
development and autonomous differentiation in ascidian embryos
have led to many investigations of the ability of one
part of the embryo to influence other cells. In an early experiment
with S. canopus embryos, Rose (1939) showed that
neural induction was necessary for the formation of the
brain. Later studies have uncovered many other cases. The
molecular nature of autonomous differentiation and induction
in ascidian embryos continues to be a very active field
of research that has been extensively reviewed (Satoh 1994,
1998, 2001, 2003; Corbo et al. 2001; Makabe et al. 2001;
Nishida 2002). In this issue, Cone and Zeller (2005) review
the evolution of developmental gene networks, while
Shimeld and Holland (2005) analyze the extensive findings
on the molecular biology of cephalochordate development
and how these findings impact our concept of the evolution
of the chordates.
Ascidian colonies are composed of numerous small zooids
embedded in a common tunic or small individual zooids
connected by stolons. Coloniality is not related to systematic
position. Colonial species are common in two orders: all
aplousobranchs and stolidobranchs from the family Styelidae.
The only colonial phlebobranchs are Perophora species and
the poorly understood Plurellidae. Ascidians use multiple
asexual methods of budding to produce colonies (reviewed
by Nakauchi 1982; Nakauchi and Kawamura 1990; Satoh
1994). Life cycles of zooids in Botryllus schlosseri colonies
show a regular alternation between budding and death by
apoptosis (Lauzon et al. 2002). Colonies grow larger by
spreading on the substrate and frequently two colonies grow
into each other. They can either fuse or reject each other
when they meet. Colony fusion and rejection has fascinated
biologists for decades and continues to be an active area of
research; in this issue, Rinkevich (2005) surveys the function
of the immune system during colony fusion. In many
stolidobranchs, such as Botryllus species, colony fusion is
genetically controlled (Scofield et al. 1982; Watanabe and
Taneda 1982; for reviews see Satoh 1994), but colonies of the
aplousobranch Diplosoma listerianum fuse indiscriminately
and the resulting colony is a chimera containing zooids of
several genotypes (Bishop and Sommerfeldt 1999).
Structure and function
Structure and function are inextricably linked and have
been extensively reviewed (Goodbody 1974; Kott 1989a;
Burighel and Cloney 1997). In this issue, Lacalli (2005) reviews
protochordate body plans and the evolution of larvae,
while Cameron (2005) reviews the morphological phylogeny
of the hemichordates. In addition, Ruppert (2005) reviews
the relationship and evolution of structural similarities between
the protochordate phyla.
Hormones are chemical signals operating to convey messages
within the organism. Most were originally discovered
in vertebrates or insects, but several are also found in protochordates
and are important in evolutionary and functional
issues. The review by Sherwood et al. (2005) on the endocrinology
of protochordates covers the known hormones and
their receptors, as well as the evolution of the endocrine system.
Protochordates have evolved nervous systems quite different
from most protostomes and the other deuterostomes.
Certain similarities exist between the protochordate and vertebrate
nervous systems, and these are crucial to understanding
evolutionary relationships. Because the central and
peripheral nervous systems of the ascidian larva have the
simplest structure in the chordate lineage, being composed
of only a few hundred cells (Nicol and Meinertzhagen 1991),
and because of their invariant cleavage pattern (Meinertzhagen
and Okamura 2001), development of this system is
under intense scrutiny (Meinertzhagen et al. 2000). In this
issue, Meinertzhagen (2005) reviews the development, structure,
and function of the larval ascidian nervous system, and
Mackie and Burighel (2005) review the adult nervous system.
Cephalochordates exhibit a very much reduced degree
of cephalization compared with vertebrates. Consequently,
the structure and function of their nervous system are proving
crucial to understanding protochordate relationships (see
review by Wicht and Lacalli 2005).
These reviews show that while we have learned a great
deal about protochordates, there is still much more work to
be done to understand this key evolutionary group of organisms.
“The study of animals is always the study of unresolved
problems, and of no group is this more true than the
protochordates” (Barrington 1965). We hope that these reviews
inspire renewed efforts and serve to focus research
into unresolved questions.
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